New antibiotics based on novel chemical scaffolds against new bacterial targets are vital to reducing the tide of tuberculosis (TB) infection worldwide. Due to the lengthy treatment requirements and the difficulty in achieving treatment compliance, there is an increasing rate of development of multi-drug resistant TB. In addition, the coinfection of TB with HIV, in areas where AIDS is epidemic, is killing 1 in 3 people with AIDS. The goal of this research project is to discover and develop novel antibiotics effective against Mycobacterium tuberculosis (Mtb). Our strategy is to screen for novel inhibitors of the DNA replication pathway, which consists of multiple essential, drug-validated targets. In Phase I, we will engineer a non-pathogenic M. smegmatis strain to build a permeable-cell DNA replication pathway screen. We will validate this screen, optimize it for high throughput, and apply it to a diverse library of 100K discrete chemical and natural product structures. Compounds that inhibit DNA replication will be confirmed in replicate assays and evaluated in profiling assays where we test the validated hits for purity and mass, potency on intact Mtb cells, specificity for inhibition of DNA replication, selectivity with respect to mammalian cells in culture, and other drug-like properties. Finally, we will attempt to identify enzyme targets against which compounds are inhibitory. We will begin with 2 essential DNA replication targets-DNA polymerase III (pol III) and DNA gyrase. Microbiotix has considerable experience with the preparation and assay of these enzymes in low G:C Gram+ bacteria. The specific aims are to (1) develop a permeable-cell, high-throughput DNA replication pathway assay using M. smegmatis; a close relative of Mtb; (2) screen a diverse compound library with the high-throughput DNA replication assay and confirm resulting hits; (3) profile confirmed hits for activity on bacterial cells, specificity for DNA synthesis inhibition, and mammalian cell cytotoxicity; and (4) construct biochemical secondary assays for pol III and gyrase from Mtb. In Phase II, we will optimize the most promising compounds to develop novel leads and candidate drugs for pre-clinical testing in animal models of infection. [unreadable] [unreadable] [unreadable]